Method of identifying or evaluating synergistic combinations of actives and compositions containing the same

ABSTRACT

A method of identifying or evaluating synergistic combinations of actives such as skin-care actives that improve the metabolism of a cell and provides an advantage over using the actives alone. The method includes contacting cells with test agents, alone and in combination, and determining a response of the cells to the test agent(s), wherein the response corresponds to a change in a metabolic indicator associated with glycolysis and a change in a metabolic indicator associated with oxidative phosphylation.

FIELD OF THE INVENTION

The present invention is directed, generally, to a method of identifyingor evaluating beneficial combinations of active ingredients for use inpersonal care compositions. More specifically, the present invention isdirected to a method of identifying or evaluating synergisticcombinations of actives that combat the effects of oxidative stress onskin cells.

BACKGROUND OF THE INVENTION

A fundamental basis for life is the need and ability of an organism togenerate energy. In humans, food is taken in and converted into chemicalcompounds such as adenosine triphospate (“ATP”) and nicotinamide adeninedinucleotide (“NAD”), which store the energy used by the cells of thebody to perform the biological processes that sustain life. Themetabolic pathways of the cells that convert the useful components offood (e.g., carbohydrates, fats and proteins) into usable energy arecomplex and may be affected by a variety of factors in ways that are notcompletely understood. Mammalian skin cells are no exception. Skin cellsare known to include a variety of different kinds of cells that functiontogether in a dynamic, complex relationship to maintain the health ofthe skin. For example, keratinocytes proliferate and differentiate toprovide continuous skin turnover. Melanocytes are known to providemelanin synthesis for skin pigmentation. And fibroblasts are known forsynthesizing the extracellular matrix and collagen, which helps maintainthe skin's thickness and elasticity. Similarly, other cells found in oraround the skin or other bodily organs, such as myocytes, stem cells,sebocytes, neurocytes, and adipocytes, all require energy derived fromcomplex metabolic pathways, which can be undesirably impacted by avariety of different factors.

There is a growing awareness of the impact of various stressors oncellular bioenergetics and the impacts on cell aging, as well as otherdiseases (e.g., cancer, neurodegenerative diseases, diabetes, andcardiovascular disorders). One theory underlying some of these effectsof altered metabolism in disease states is the Free Radical Theory ofaging. Namely, that exposure of mammalian cells to reactive oxygenspecies (“ROS”) causes damage to cellular structures and organelles suchas the mitochondria. ROS are highly reactive molecules that containoxygen (e.g., oxygen ions and peroxides). ROS are formed naturallywithin cells as a natural byproduct of the normal metabolism of oxygenand play a role in cell signaling and homeostasis. However, when a cellis exposed to a stressor such as heat or UV radiation, ROS levels canincrease, and in some instances dramatically.

As the damage caused by ROS accumulates over time, it causes more andmore oxidative stress at the cellular level that ultimately may lead totissue damage and/or organ dysfunction. One effect of oxidative stresson cells is a diminished capacity of cellular bioenergetics, which canlead to reduced levels of ATP and/or NAD. This may be particularlyproblematic for human skin because the oxidative stress on human skincells may manifest as visible signs of aging. Further, environmentalstressors such as ultraviolet radiation (“UV”) and pollutants (e.g.,cigarette smoke, car exhaust, ozone) can lead to heightened levels ofROS production. Over time, this may result in noticeable changes in theskin's structure and morphology (e.g., “photodamage”) and, to a moreextreme degree, skin carcinomas.

Human skin cells defend against ROS by using redox regulators such asglutathione and NAD as well as various enzymes that can neutralize ROS.However, these defenses can be overwhelmed by the elevated spike fromstressor-induced ROS, leading to not just acute but also chronicalterations in energy homeostasis and metabolism efficiencies, causingoverall cellular dysfunction. To complicate matters, the variety ofcells types associated with human skin and the complexity of themetabolic pathways of these cells makes it difficult to identifysuitable compounds to help combat the anti-aging effect associated withexposure to a particular oxidative stressor or combination of stressors.Certain oxidative stressors affect different types of cells and/ormetabolic pathways differently. This makes it difficult to select asuitable skin care active or combination of actives (whose affect mayalso vary depending on type of cell or metabolic pathway) that combatthe undesirable affects of a particular stressor or stressors. Whileidentifying actives that prevent, reduce and/or reverse the effects ofoxidative stress on a cell is desirable, it is even more desirable toidentify combinations of actives that act together to provide anadvantage over using a single active. This is sometimes referred to assynergy. Identifying skin-care actives that work alone to combat theeffects of oxidative stress can be difficult, but quickly and/orefficiently identifying skin-care actives that work in synergy can beformidable. Thus, there is a long felt need for a method of identifyingsynergistic combinations of skin-care actives that combat theundesirable metabolic effects associated with various oxidativestressors, especially common environmental stressors.

As an initial step in finding synergistic combinations of skin-careactives, a method capable of detecting the changes in cellularmetabolism caused by stressors and actives must be identified. Variousmethods are known for evaluating the energy making processes of cells.For example, Clark-type electrode probes are known for measuring oxygenconsumption. The Clark electrode provides kinetic information (i.e.,rates of response) but introduces artifact (i.e., some undesirableand/or extraneous factor that influences the results of a test) by itscontinuous consumption of oxygen, presenting a decreasing oxygenpressure to the cells or isolated mitochondria in the measurementchamber. Although oxygen consumption may provide an indication ofmitochondrial function, it only measures one component of cellularbioenergetics and does not provide an assessment of other metabolicpathways that contribute to bioenergetic equilibrium, namely glycolysis.

Another conventional method for assessing cellular energy production isby measuring the amount of ATP in a cell. Luminescent ATP assay kits arecommercially available for quantifying total energy metabolism. ATPassays are known to be relatively sensitive but they may not be an idealmetric of mitochondrial function as cells strive to maintain aparticular ATP budget and will adjust metabolism accordingly. Thus,alterations in ATP levels are usually only detectable duringpathophysiological changes. In addition, ATP assays are destructive(i.e., the cells are destroyed in order to measure the amount of ATP)and they lack kinetic information. Further, artifact has been reportedfrom residual ATP present in dying or dead cells. And like theClark-electrode assay, ATP cannot determine the relative contribution ofdifferent metabolic pathways to total ATP yield.

Still another convention method for assessing cell energetics is withcommercially available MTT/XTT or alamarBlue™ kits. While these kits mayprovide a relatively simple way to assess cell health, they are not assensitive as ATP assays. In addition, they have been reported tointroduce error through cell toxicity, the very parameter they aresupposed to be measuring. Further, both assays are destructive and donot provide kinetic information.

Accordingly, it would be desirable to provide a method of identifyingand/or evaluating beneficial combinations of actives that reduce,prevent and/or reverse the undesirable oxidative stress on skin cells.It would also be desirable to provide a skin care composition thatincludes skin-care actives identified by the foregoing method. It wouldfurther be desirable to provide a method of treating skin damaged by theoxidative stress effects of a particular stressor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1 b and-1C are illustrations of the oxygen consumption rate ofkeratinocytes.

FIG. 2 is an illustration of the oxygen consumption rate ofkeratinocytes.

FIGS. 3A, 3B and 3C are illustrations of the extracellular acidificationrate of keratinocytes.

FIG. 4 is an illustration of the extracellular acidification rate ofkeratinocytes.

FIGS. 5A, 5B, 5C, 5D and 5E are illustrations of the oxygen consumptionrate of fibroblasts.

FIG. 6 is an illustration of the oxygen consumption rate of fibroblasts.

FIGS. 7A, 7B, 7C, 7D and 7E are illustrations of the extracellularacidification rate of fibroblasts.

FIG. 8 is an illustration of the extracellular acidification rate offibroblasts.

FIG. 9 is an illustration of the extracellular acidification rate offibroblasts.

FIG. 10 is an illustration of the oxygen consumption rate offibroblasts.

SUMMARY OF THE INVENTION

In order to provide a solution to the aforementioned problems, disclosedherein is a method of identifying or evaluating a synergisticcombination of actives for use in a personal care composition. Themethod comprises: (a) providing first, second and third test vessels,each test vessel comprising cells disposed in a medium; (b) contactingthe cells in the first test vessel with a first test agent; (c)non-lethally detecting a metabolic indicator associated with each of aglycolysis metabolic pathway and an oxidative phosphorylation metabolicpathway of the cells in the first test vessel to provide a response ofeach metabolic pathway to the first test agent; (d) contacting the cellsin the second test vessel with a second test agent; (e)non-lethallydetecting the a metabolic indicator associated with each of theglycolysis and oxidative phosphorylation metabolic pathways of the cellsin the second test vessel to provide a response of each metabolicpathway to the second test agent; (f) contacting the cells in the thirdtest vessel with the first and second test agents in combination; (g)non-lethally detecting the a metabolic indicator associated with each ofthe glycolysis metabolic pathway and the oxidative phosphorylationmetabolic pathway of the cells in the third test vessel to provide aresponse of each metabolic pathway to the first and second test agentsin combination; and (h) identifying the combination of the first andsecond compositions as a beneficial synergistic combination of activeswhen the response for at least one of the glycolysis and oxidativephosphorylation metabolic pathways responses in (g) is an improvementover the corresponding response of the same metabolic pathway in (c) or(e).

In some embodiments, the method may comprise: (a) providing a first testvessel comprising a first type of cells disposed in a first medium and asecond test vessel comprising a second type of cells disposed in asecond medium; (b) providing a basal value for each of a glycolysismetabolic pathway and an oxidative phosphorylation metabolic pathway ofthe first type of cells and the second type of cells; (c) contacting thefirst and second types of cells with a first test agent and a secondtest agent in combination; (d) non-lethally detecting a metabolicindicator associated with each of the glycolysis and oxidativephosphorylation metabolic pathways of the first and second types ofcells to provide a response of each metabolic pathway to the first andsecond test agents in combination; and (e) identifying the combinationof the first and second test agents as a beneficial combination ofactives when the responses of at least one of the glycolysis andoxidative phosphorylation metabolic pathways of each of the first andsecond types of cells indicates an improvement over the correspondingbasal value.

In some embodiments, the method may comprise: (a) providing first,second and third test vessels, each test vessel comprising cellsdisposed in a medium; (b) contacting the cells in the first test vesselwith a first test agent; (c) non-lethally detecting a metabolicindicator associated with each of a glycolysis metabolic pathway and anoxidative phosphorylation metabolic pathway of the cells in the firsttest vessel to provide a response of each metabolic pathway to the firsttest agent; (d) contacting the cells in the second test vessel with asecond test agent; (e) non-lethally detecting a metabolic indicatorassociated with each of the glycolysis and oxidative phosphorylationmetabolic pathways of the cells in the second test vessel to provide aresponse of each metabolic pathway to the second test agent; (f)contacting the cells in the third test vessel with the first and secondtest agents in combination; (g) non-lethally detecting a metabolicindicator associated with each of the glycolysis metabolic pathway andthe oxidative phosphorylation metabolic pathway of the cells in thethird test vessel to provide a response of each metabolic pathway to thefirst and second test agents in combination; and (h) identifying thecombination of the first and second test agents as a beneficialcombination of actives when the response for at least one of theglycolysis and oxidative phosphorylation metabolic pathways in (g) isnot worse than the response of the same metabolic pathway in (c) and (e)and at least one of the first and second test agents provides anon-metabolic benefit over the other of the first and second testagents.

In some embodiments, there is disclosed a personal care compositioncomprising a synergistic combination of skin-care actives, thecomposition comprising: (a) a dermatologically acceptable carrier; (b) asafe and effective amount of niacinamide as a first active; and (c) asafe and effective amount of tocoquinone as a second active, wherein thesafe and effective amount of niacinamide and tocoquinone in combinationimproves at least one of glycolysis and oxidative phosphorylationmetabolism of a skin cell suffering from oxidative stress as a result ofexposure to a stressor and provides a synergistic advantage over usingthe actives alone.

DETAILED DESCRIPTION OF THE INVENTION

All percentages are by weight of the personal-care composition, unlessotherwise specified. All ratios are weight ratios, unless specificallystated otherwise. All numeric ranges are inclusive of narrower ranges;delineated upper and lower range limits are interchangeable to createfurther ranges not explicitly delineated. The number of significantdigits conveys neither limitation on the indicated amounts nor on theaccuracy of the measurements. All measurements are understood to be madeat about 25° C. and at ambient conditions, where “ambient conditions”means conditions under about one atmosphere of pressure and at about 50%relative humidity.

DEFINITIONS

“Additive effect” means that the effect provided by a combination ofactives is equal to or substantially equal to the sum of theirindividual effects. For example, an additive effect would bedemonstrated when a first active, which provides a 10% improvement inoxygen consumption rate when used alone, and a second active, whichprovides a 20% improvement in oxygen consumption rate when used alone,provide a 30% improvement to oxygen consumption rate when used incombination. A “more than additive effect,” in this example, would bedemonstrated when the combination of the first and second activesimproves oxygen consumption rate by more than 30%.

“Cosmetic” means providing a desired visual effect on an area of thehuman body. The visual cosmetic effect may be temporary, semi-permanent,or permanent. Some non-limiting examples of “cosmetic products” includeproducts that leave color on the face, such as foundation, mascara,concealers, eye liners, brow colors, eye shadows, blushers, lip sticks,lip balms, face powders, solid emulsion compact, and the like.

“Dermatologically acceptable” means that the compositions or componentsthereof being described are suitable for use in contact with human skinwithout undue toxicity, incompatibility, instability, allergic response,and the like.

“Different types of cells” means cells that differ from one another intheir intended biological function. Examples of different types ofcells, with respect to one another, include, without limitation,fibroblasts, keratinocytes, melanocytes, myoctyes, sebocytes andadipocytes.

“Disposed” means an element is positioned in a particular place relativeto another element.

“Non-lethal,” means that a test procedure in not intended to kill ordestroy the cells being tested or observed. For example, non-lethallydetecting a metabolic indicator means that at least 75% of the cells areviable after the detection (e.g., 80%, 85%, 90%, 95% or even up to 99%or more of the cells remain viable). Ideally, 100% of the cells areviable after a non-lethal test, but it is to be appreciated that thedeath or destruction of some cells may be unavoidable and/or unrelatedto the test.

“Oxidative Stressor” means an environmental element that causes theformation of undesirable reactive oxygen species in a cell. Somenon-limiting examples of oxidative stressors include ultravioletradiation, cigarette smoke, ozone, engine exhaust, diesel exhaust, smog,surfactants, and radiation from a computer monitor or television.

“Personal-care composition” means a composition suitable for topicalapplication on mammalian skin. The personal care compositions herein maybe used in skin-care, cosmetic, and hair-care products; non-limitinguses of which include antiperspirants, deodorants, lotions (e.g. handlotion and body lotion), skin-care products (e.g., face and necklotions, serums, sprays), sunless tanners, cosmetics (e.g., foundation,concealer, blush, lipstick, lip gloss), depilatories, shampoos,conditioning shampoos, hair conditioners, hair dyes, body washes,moisturizing body washes, shower gels, skin cleansers, cleansing milks,hair and body washes, in-shower body moisturizers, pet shampoos, shavingpreparations, after-shaves, razor moisturizing/lubricating strips, razorshave-gel bars, bar soaps, cleansing products, feminine-care products,oral-care products, and baby-care products. The methods of using any ofthe aforementioned compositions are also included within the meaning ofpersonal-care composition.

“Regulate a skin condition” means maintaining skin appearance and/orfeel with little to no degradation in appearance and/or feel.

“Safe and effective amount” means an amount of a compound or compositionsufficient to significantly induce a positive benefit, preferably apositive skin or feel benefit, including independently or incombinations the benefits disclosed herein, but low enough to avoidserious side effects, i.e., to provide a reasonable benefit to riskratio, within the scope of sound judgment of the skilled artisan.

“Skin” means the outermost protective covering of mammals that iscomposed of skin cells such as keratinocytes, fibroblasts andmelanocytes. Skin includes an outer epidermal layer and an underlyingdermal layer. Skin may also includes hair and nails as well as othertypes of cells commonly associated with skin such as, for example,myocytes, Merkel cells, Langerhans cells, macrophages, stem cells,sebocytes, nerve cells and adipocytes.

“Skin-care” means regulating and/or improving a skin condition. Somenonlimiting examples include improving skin appearance and/or feel byproviding a smoother, more even appearance and/or feel; increasing thethickness of one or more layers of the skin; improving the elasticity orresiliency of the skin; improving the firmness of the skin; and reducingthe oily, shiny, and/or dull appearance of skin, improving the hydrationstatus or moisturization of the skin, improving the appearance of finelines and/or wrinkles, improving skin exfoliation or desquamation,plumping the skin, improving skin barrier properties, improve skin tone,reducing the appearance of redness or skin blotches, and/or improvingthe brightness, radiancy, or translucency of skin. Some nonlimitingexamples of “skin-care products” include skin creams, moisturizers,lotions, and body washes.

“Skin-care composition” means a composition that regulates and/orimproves skin condition.

“Skin-care active” mean a compound or a combination of two or morecompounds that, when applied to skin, provide an acute and/or chronicbenefit to skin or a type of cell commonly found therein. Skin-careactives may regulate and/or improve skin or its associated cells (e.g.,improve skin elasticity; improve skin hydration; improve skin condition;and improve cell metabolism).

“Synergy” and variations thereof mean that a combination of two or moreactives improves the metabolism of a cell (e.g., glycolysis or oxidativephosphorylation) and provides an advantage (e.g., biological and/oreconomic) over using the actives alone. In some embodiments, synergy maybe demonstrated by a combination of actives acting together to improvethe metabolism of a cell beyond what the combination would normally beexpected to provide. For example, if a combination of actives isexpected to provide an additive effect, but instead provides a more thanadditive effect, the combination may be considered synergistic.Similarly, if a combination of actives is expected to provide a lessthan additive effect, but instead provide an additive effect, thecombination may be considered synergistic. In some embodiments, synergymay be demonstrated by a combination of actives acting on differentmetabolic pathways (e.g., glycolysis and oxidative phosphorylation) toimprove overall cell metabolism more than either active working alone.In some embodiments, synergy may be demonstrated by a combination ofactives that each act on a different type of cell (e.g., keratinocytesand fibroblasts) to improve the metabolism of that type of cell, therebyimproving the overall health of the skin. In some embodiments, synergymay be demonstrated by a combination of actives that do not inhibit oneanother with respect to improving the metabolism of a cell and/ormetabolic pathway. In some embodiments, synergy may be demonstrated by acombination of actives wherein all the actives in the combinationprovide a metabolic benefit and at least one of the actives provides anon-metabolic benefit (e.g., economic, formulation, regulatory and/orsafety benefit).

“Topical application” means to apply or spread the compositions of thepresent invention onto the surface of the keratinous tissue.

As humans age, damage from external and internal stressors on the cellsof the body accumulates (i.e., oxidative stress), which may lead todecreased efficiency and function of tissue and organs. It is notuncommon for oxidative stress to manifest as a reduction in the abilityof the cells to produce energy. Skin-care actives that reduce, stop oreven reverse the effects of oxidative damage to skin are known. Butidentifying new and/or better skin-care actives is difficult due to thecomplexity of the metabolic pathways of a cell and/or the number ofdifferent types of cells found in skin. Surprisingly, it has been foundthat certain oxidative stressors impact the bioenergetics of certaintypes of skin cells in previously unappreciated ways. This new learningmay be used to identify beneficial combinations of skin-care activesthat combat the undesirable metabolic effects of oxidative stress onskin cells, which in turn may lead to improved skin-care compositions,more efficient methods of identifying combinations of actives and/or amore holistic approach to skin care. Accordingly, the novel methodherein provides a convenient and accurate way to identify beneficialcombinations of skin-care actives that act to combat the undesirablemetabolic effects of oxidative stressors in ways that were previouslyunappreciated.

While some examples herein may be directed to skin cells such askeratinocytes and fibroblasts in conjunction with UV radiation, it is tobe appreciated that the method may be adapted to great advantage for usewith any type of cell in conjunction with any stressor, as desired.

Two key metabolic pathways for mammalian cells to produce energy are theoxidative phosphorylation (“oxphos”) pathway and the glycolysis pathway.Both pathways are necessary to maintain a healthy energy balance withinmost mammalian cells. Oxidative phosphorylation involves the transfer ofelectrons from electron donors to electron acceptors such as oxygen inredox reactions, which results in the release of energy that is used toform ATP. In mammals, the redox reactions are carried out by a series ofprotein complexes within the mitochondria membrane, and the linked setsof proteins are called electron transport chains. The energy released byelectrons flowing through this electron transport chain is used totransport protons across the mitochondrial membrane in a process calledchemiosmosis, which generates potential energy in the form of a pHgradient and an electrical potential across this membrane. An enzymecommonly known as ATP synthase allows the potential energy to be used togenerate ATP. Because the oxidative phosphorylation pathway uses oxygento generate ATP, the rate at which a cell consumes oxygen may be used asa metabolic indicator of the cell. That is, the oxygen consumption rateof the cell may be directly correlated to energy production by the cellvia oxidative phosphorylation. Additionally or alternatively, the carbondioxide production rate may also be used as a metabolic indicator, sincecarbon dioxide is a by-product of cellular metabolism. A higher oxygenconsumption rate or carbon dioxide production rate may indicate anincrease in energy production from the oxidative phosphorylationpathway, and thus an improvement in the metabolism and/or health of acell. Conversely, a lower oxygen consumption rate or carbon dioxideproduction rate may indicate a decrease in oxphos metabolism. As aresult, it would desirable to identify and/or evaluate compounds thatreduce, prevent and/or reverse the decrease in oxphos metabolism causedby oxidative stressors, especially common environmental stressors.

Glycolysis is the metabolic pathway that converts glucose into pyruvate.The free energy released in this process is used to form ATP and NADH(reduced NAD). Glycolysis is a definite sequence of ten reactionsinvolving ten intermediate compounds (one of the steps involves twointermediates) that typically occurs in the cytosol of the cell. Theintermediates provide entry points to glycolysis. For example, mostmonosaccharides such as fructose, glucose, and galactose, can beconverted to one of these intermediates. The intermediates may also bedirectly useful. For example, the intermediate dihydroxyacetonephosphate (DHAP) is a source of the glycerol that combines with fattyacids to form fat. A by-product of glycolysis is lactic acid, which canform a lactate anion plus a proton in solution. Thus, lactic acid,lactate or proton concentration can be used as a metabolic indicator ofglycolysis. That is, a change in extracellular pH or extracellularacidification rate may be directly correlated to energy production bythe cell via the glycolysis pathway. A higher extracellularacidification rate may indicate an increase in energy production via theglycolysis pathway, and thus an improvement in the metabolism and/orhealth of a cell. Conversely, a lower extracellular acidification ratemay indicate a decrease in glycolysis metabolism. As a result, it woulddesirable to identify and/or evaluate compounds that reduce, preventand/or reverse the decrease in gycolysis metabolism caused by oxidativestressors, especially common environmental stressors.

Keratinocytes

Keratinoctyes are generally recognized as the predominant cell type inthe epidermis, typically constituting about 95% of the cells foundthere. Keratinocytes are formed by differentiation from epidermal stemcells residing in the lower part of the stratum basale layer of theepidermis. Keratinocytes divide and differentiate as they move upwardthrough the layers of the epidermis (e.g., stratum spinosum and stratumgranulosum) to eventually become corneocytes in the stratum corneum.During the differentiation process, keratinocytes produce more and morekeratin (“cornification”) and eventually permanently withdraw from thecell cycle to form the corneocytes that make up the hard outer layer ofthe stratum corneum. Corneocytes are eventually shed off throughdesquamation as new cells come in. When oxidative stress reduces themetabolism of keratinocytes, the rate at which the keratinocytes divideand differentiate may be reduced or even halted. This, in turn, mayreduce the rate at which lost corneocytes are replaced in the stratumcorneum and ultimately lead to an undesirable decrease in the barrierproperties of the skin. Thus, it may be desirable to identifysynergistic combinations of skin-care actives that reduce, preventand/or reverse the undesirable metabolic effects of oxidative stressfrom certain stressors (e.g., UV-A, UV-B, cigarette/tobacco smoke, smog,ozone, engine exhaust, volatile organic compounds) on keratinocytes.

It is well known that exposing skin to UV radiation can cause oxidativestress to skin cells. However, before now, it was not fully appreciatedhow particular types of skin cells react metabolically to differentwavelengths of UV radiation, or how the metabolic reaction of aparticular type of skin cell to UV radiation may change with time afterexposure to the UV radiation. It has now been found that the oxphos andglycolysis metabolic pathways of keratinocytes respond differently toUV-B radiation (i.e., electromagnetic radiation in the wavelength rangeof 315-280 nm). In particular, it has been found that exposure ofkeratinocytes to UV-B radiation causes a decrease in metabolic activityin the oxphos pathway, but does not appear to have the same effect onthe glycolysis pathway. Consequently, it may be desirable to identifysynergistic combinations of skin-care actives that reduce, preventand/or reverse the undesirable metabolic effects of UV-B radiation onkeratinocytes. It may also be desirable to identify beneficialcombinations of actives that reduce, prevent and/or reverse theundesirable metabolic effects of the same or different oxidativestressors and/or ROS on keratinocytes and at least one other type ofcell commonly found in skin (e.g., fibroblasts, melanocytes, adipocytes,stem cells, sebocytes and neurocytes) to improve the overall health ofthe skin.

It has also been found that keratinocytes exhibit a previouslyunappreciated metabolic response to UV-B radiation at a particular doseand/or time after exposure. This discovery provides unique insight intoscreening for synergistic combination of skin-care actives to combat theeffects of oxidative stress on keratinocytes from UV-B radiation. Inparticular, it is now known that between 5 milliJoules per squarecentimeter (“mJ/cm²”) and 50 mJ/cm² (e.g., from 7.5-40 mJ/cm², 10-30mJ/cm², or even 15-30 mJ/cm²) of UV-B radiation provides sufficientenergy to induce a measurable metabolic response in the oxphos and/orglycolysis pathway of keratinocytes, but does not kill thekeratinocytes. Further, in some instances it can be important to detectthe desired metabolic indicator at least 1 hour after exposure of thekeratinocytes to a stressor, but typically no more than 72 hours afterexposure (e.g., from 2 to 24 hours; 3-23 hours; 4-22 hours; 5-21 hours;6-20 hours; 7-19 hours; 8-18 hours; 9-17 hours; 10-16 hours; 11-15hours; or even 12-14 hours). If the metabolic indicator is detected toosoon, the cells may not have sufficient time to fully respond to thestressor. On the other hand, if too much time passes after exposure, aresponse may be missed (e.g., if the metabolism of the cell returns tothe basal value). Further, it is now known that, in some instances, thekinetic data observed at particular times can provide important insightsinto the responses of keratinocytes to oxidative stressors and/or ROS,which may not be apparent when using a conventional static detectionmethod (e.g., ATP assay).

In some instances, the method herein comprises exposing a plurality ofkeratinocytes to a stressor such as UV-B radiation and/or a ROS such ashydrogen peroxide and then contacting the keratinocytes with two or moretest agents, alone and in combination. The metabolic responses of thekeratinocyte to the stressor and test agents are obtained by detecting ametabolic indicator corresponding to each of the oxphos and glycolysismetabolic pathways. The metabolic responses are detected in real time ina controlled environment, and the oxphos and glycolysis indicators areobtained from the same cells at the same time. In some embodiments, itmay be desirable to provide a basal value for each of the oxphos andglycolysis metabolic pathways of the keratinocyte and compare themetabolic responses to the basal value and/or each other to determinethe response of the metabolic pathway to the stressor and/or testagents. “Basal value” means the value of the metabolic indicator at anormal resting state prior to exposure of the cell to a stressor or testagent. The basal value of a cell may be provided by measuring themetabolic indicators and/or by consulting the scientific literatureand/or other suitable sources. However, measuring the basal value may bepreferred since it is well known that the basal metabolic values ofcells may vary due to a variety of environmental and intrinsic factors.

FIGS. 1A, 1B and 1C illustrate kinetic data obtained by detecting theoxygen consumption rate (“OCR”) of keratinocytes after exposure to UV-Bradiation (312 nanometers). The keratinocytes illustrated in the figuresare primary keratinocytes obtained from Gibco Life Technologies, GrandIsland, N.Y., USA. The cells were prepared and tested according to themethod described in the Test Method below. The upper plots 30A, 30B and30C in each of FIGS. 1A, 1B and 1C illustrate the basal OCR values ofthe keratinocytes (i.e., OCR with no UV-B exposure). The lower plots35A, 35B and 35C of each of FIGS. 1A, 1B and 1C illustrate the responseOCR values of the keratinocytes after exposure to UV-B radiation dosesof 7.5, 15 and 30 mJ/cm², respectively. It is to be appreciated thatproviding the proper dose (i.e., 7.5, 15 or 30 mJ/cm²) can depend on avariety of well-known factors (e.g., dose level desired, age of lightbulb, type of light bulb, whether the instrument is warm), and it iswell within the ability of one of ordinary skill to provide the desireddose of UV radiation. The control plots 37A, 37B and 37C illustrate thecontrol OCR values measured on a well that did not contain anykeratinocytes, but included the same medium as the other wells. Thecontrol values enable correction of the basal and/or response values forany background effect that may be present. The first data point in eachplot is taken approximately one hour after exposure to UV-B radiation.As illustrated in FIGS. 1A, 1B and 1C, the response OCR values of thekeratinocytes were generally lower than the corresponding basal OCRvalues. It is believed, without being limited by theory, that the dataillustrated in FIGS. 1A, 1B and 1C indicate that UV-B radiationdecreases the oxphos metabolism of keratinoctyes. Consequently, it wouldbe desirable to identify synergistic combinations of skin-care activesthat combat the undesirable effects of a stressor such as UV-B radiationon the oxphos metabolic pathway of keratinocytes, for example, by actingdirectly (i.e., cause an improvement even without the presence of astressor or ROS) and/or indirectly (e.g., cause an improvement only whenthe cell is exposed a stressor or ROS) on the oxphos and/or glycolysismetabolic pathways of the keratinocytes and, optionally, another type ofcell.

FIG. 2 illustrates the keratinocyte OCR response 24 hours after exposureto UV-B radiation (312 nanometers). The 24-hour values illustrated inFIG. 2 were obtained by exposing the keratinocytes to UV-B radiation andthen incubating the plate at 37° C. and 5% CO₂ for 24 hours. Twenty-fourhours after UV exposure, the cells were analyzed according to the methoddescribed below. The 24-hour basal value 40 is shown at the far leftside of the chart 49. Immediately to the right of the basal value 40 isthe 24-hour response OCR value 41 for a 7.5 mJ/cm² dose of UV-Bradiation, followed by the 24-hour response OCR value 42 for a 15 mJ/cm²dose. And at the far right of the chart 49 is the 24-hour response OCRvalue 43 for a 30 mJ/cm² dose. As illustrated in FIG. 2, thekeratinocytes continued to show a reduction in OCR 24 hours afterexposure to UV-B doses of 7.5, 15 and 30 mJ/cm². Thus, it may beimportant to measure the keratinocyte oxphos response up to 24-hours ormore (e.g., 48 or 72 hours) after exposure to UV-B, particularly whenscreening for synergistic combinations of skin-care actives that reduce,prevent and/or reverse the negative effects of UV-B on kertainocyteoxphos metabolism.

FIGS. 3A, 3B and 3C illustrate kinetic data obtained by detecting theextra cellular acidification rate (“ECAR”) of keratinocytes afterexposure to UV-B radiation (312 nanometers). The keratinocytes areprimary keratinocytes obtained in the same way as described above withregard to FIGS. 1A, 1B and 1C, and the test was performed according tothe method described in the Test Methods section below. The upper plots65A, 65B and 65C in each of FIGS. 3A, 3B and 3C illustrate the responseECAR values of the keratinocytes after exposure to UV-B radiation dosesof 7.5, 15 and 30 mJ/cm², respectively. The lower plot 60A, 60B and 60Cillustrates the basal ECAR values of the keratinocytes (i.e., no UV-Bexposure). The control plots 67A, 67B and 67C illustrate the controlECAR values measured on a well that did not contain any keratinocytes,but included the same medium as the other wells. As illustrated in FIGS.3A, 3B and 3C, the control values 67A, 67B and 67C did not show anymeasurable change over time, whereas the response ECAR values of thekeratinocytes were generally higher than the corresponding basal ECARvalues. A statistical analysis of the ECAR response data was conductedusing a 1-way analysis of variance (“ANOVA”) with a Dunnett'scorrection, and the statistical analysis showed that the there was nostatistically significant change in the ECAR response values 65A, 65Band 65C relative to the corresponding basal values 60A, 60B and 60C.

FIG. 4 illustrates the keratinocyte ECAR response 24 hours afterexposure to UV-B radiation (312 nanometers). The 24-hour valuesillustrated in FIG. 4 were obtained by exposing the keratinocytes toUV-B radiation and then incubating the plate at 37° C. and 5% CO₂ for 24hours. Twenty-four hours after UV exposure, the cells were analyzedaccording to the method described below. The 24-hour basal ECAR value 70is shown at the far left side of the chart 79. Immediately to the rightof the basal value 70 is the 24-hour response ECAR value 71 for a 7.5mJ/cm² dose of UV-B radiation, followed by the 24-hour response ECARvalue 72 for a 15 mJ/cm² dose. And at the far right of the chart 79 isthe 24-hour response ECAR value 73 for a 30 mJ/cm² dose. A statisticalanalysis of the 24-hour ECAR response data was conducted using a 1-wayANOVA with a Dunnett's correction. The analysis indicated that the therewas a statistically significant change in the ECAR response valuesrelative to the basal value. Thus, the amount of time that passes afterexposure of keratinocytes to a stressor such as UV-B radiation may be animportant factor to consider when screening for synergistic combinationsof skin-care actives that reduce, prevent and/or reverse the negativeeffects of UV-B on kertainocyte glycolysis metabolism. In particular, itmay be desirable to wait more than 2 hours after exposure to as stressorsuch UV-B radiation (e.g., 4, 6, 8, 10, 12, 14, 16, 18, 20, 24, 48, or72 hours) to detect a metabolic indicator of glycolysis inkeratinocytes.

Fibroblasts

In some instances, it may be desirable to identify synergisticcombinations of skin-care actives that reduce, prevent and/or reversethe undesirable effects of oxidative stress from certain stressors onfibroblasts and, optionally, other types of cells commonly found inskin. Fibroblasts are found in the dermal layer of the skin and thehypodermal (i.e., sub-cutaneous) layer, and are generally recognized asthe cells that synthesize the extracellular matrix (“ECM”) and collagento provide the structural framework for the tissues of mammals. The ECMand collagen help cushion the body from stress and strain by providingtensile strength and elasticity to the skin Fibroblasts also play animportant role in wound healing. When oxidative stress reduces themetabolism of fibroblasts, the body's ability to synthesize collagen andthe ECM may be reduced resulting in saggy, thinner looking skin. And theability of the body to heal wounds may be impeded.

As mentioned previously, it has not been fully appreciated howparticular types of skin cells react metabolically to differentwavelengths of UV radiation, or that the metabolic reaction of aparticular type of skin cell to UV radiation may change with time afterexposure to the UV radiation. This adds uncertainty and difficulty to aprocess of identifying suitable skin-care actives. Surprisingly, it hasbeen found that the oxphos and glycolysis metabolic pathways offibroblasts respond differently to UV-A radiation. While most of theUV-B radiation that contacts the skin is absorbed or reflected by thekeratinocytes, longer wavelength UV-A radiation can penetrate throughthe epidermis and damage the underlying fibroblasts and other cellscommonly found in the dermis and hypodermis. In particular, it has beenfound that exposure of fibroblasts to UV-A radiation that exceeds athreshold energy level causes a decrease in metabolic activity in theoxphos pathway. Exposure of fibroblasts to UV-A radiation also mayresult in a decrease in metabolic activity in the glycolysis pathway. Inaddition, both metabolic pathways may exhibit changes in their responsesat different energy levels and times after exposure. This discoveryprovides unique insight important for identifying synergisticcombinations skin-care actives that combat the effects of oxidativestressors such as UV-A on fibroblasts. For example, it is now known thatbetween 1 and 50 Joules per square centimeter (“J/cm²”) of UV-Aradiation may provide sufficient energy to induce a measurable metabolicresponse in the oxphos and/or glycolysis pathway, but does not generallykill the fibroblasts. Suitable ranges of UV-A radiation include between5 and 40, 10 and 30, or even about 20 J/cm2. Further, it also now knownthat in some instances it can be important to detect the metabolicindicator at least 1 hour after exposure of the fibroblasts to astressor, but typically not more than 24 hours after exposure (e.g.,from 2 to 24 hours; 3-23 hours; 4-22 hours; 5-21 hours; 6-20 hours; 7-19hours; 8-18 hours; 9-17 hours; 10-16 hours; 11-15 hours; or 12-14hours). If the metabolic indicator is detected too soon, the cells maynot have sufficient time to fully respond to the stressor. On the otherhand, if too much time elapses, an important transient response to thestressor may not be detected. Further, it is now known that, in someinstances, the kinetic data observed at particular times can provideimportant insights into the responses of fibroblasts to oxidativestressors and/or ROS, which may not be apparent when using aconventional static detection method (e.g., ATP assay).

In some embodiments, the method herein includes exposing a plurality offibroblasts to a stressor such as UV-A radiation and/or an ROS such ashydrogen peroxide and then contacting the fibroblasts with two or moretest agents alone and in combination. The metabolic responses of thefibroblasts to the stressor and/or the test agents may be obtained bydetecting a metabolic indicator corresponding to each of the oxphos andglycolysis metabolic pathways. The metabolic responses are detected inreal time in a controlled environment, and the oxphos and glycolysisindicators are obtained from the same cells at the same time. In someembodiments, it may be desirable to provide a basal value for at leastone of the oxphos and glycolysis metabolic pathways of the fibroblasts,and compare the metabolic responses to the basal value and/or each otherto determine the response of the metabolic pathway to the stressorand/or test agent.

FIGS. 5A, 5B, 5C, 5D and 5E illustrate the kinetic data obtained bydetecting the oxygen consumption rate of fibroblasts after exposure toUV-A radiation (365 nm). The fibroblasts are dermal fibroblasts obtainedfrom ATCC, Bethesda, Md. (BJ cell line). The cells were prepared andtested according to the method described in more detail below. The upperplots 90A, 90B, 90C, 90D and 90E in each of FIGS. 5A, 5B, 5C, 5D and 5Eillustrate the basal OCR values of the fibroblasts (i.e., the OCR withno UV-A exposure). The lower plots 95A, 95B, 95C, 95D and 95E of each ofFIGS. 5A, 5B, 5C, 5D and 5E illustrate the response OCR values of thefibroblasts after exposure to UV-A radiation doses of 1, 5, 10, 20 and30 J/cm², respectively. The control plots 97A, 97B, 97C, 97D and 97Eillustrate the control OCR values measured on wells that did not containany fibroblasts, but included the same medium as the other wells. Thecontrol value enables correction of the basal values and response valuesfor any background effect that may be present. As illustrated in FIGS.7A5A, 5B, 5C, 5D and 5E, the response OCR values of the fibroblasts weregenerally lower than the corresponding basal OCR values. Thus, it isbelieved, without being limited by theory, that the data illustrated inFIGS. 5A, 5B, 5C, 5D and 5E indicate that UV-A radiation, at doses ofgreater than 1 J/cm², may decrease the oxphos metabolism of fibroblasts.Consequently, it would be desirable to identify synergistic combinationsof skin-care actives that combat the undesirable effects of a stressorsuch as UV-A radiation on the oxphos metabolic pathway of fibroblasts,for example, by acting directly (i.e., cause an improvement even withoutthe presence of a stressor or ROS) and/or indirectly (e.g., cause animprovement only when the cell is exposed a stressor or ROS) on theoxphos and/or glycolysis metabolic pathways of the fibroblasts and,optionally, another type of cell.

FIG. 6 illustrates fibroblast OCR responses at 24 hours after exposureto UV-A radiation (365 nm). The 24-hour values illustrated in FIG. 6were obtained by exposing the fibroblasts to UV-A radiation and thenincubating the plate at 37° C. and 5% CO₂ for 24 hours. Twenty-fourhours after exposure to UV-A, the cells were analyzed according to themethod described below. The 24-hour basal OCR value 80 is shown at thefar left side of the chart 89. Immediately to the right of the basalvalue 80 is the 24-hour response OCR value 81 for a 1 J/cm² dose of UV-Aradiation, followed by the 24-hour response OCR value 82 for a 5 J/cm²dose. And at the far right of the chart 89 is the 24-hour response OCRvalue 83 for a 10 J/cm² dose. As illustrated in FIG. 6, the fibroblastsappear to show a reduction in OCR after 24 hours only at a dose of 10J/cm². Thus, it may be important to measure the fibroblast oxphosresponse up to 24-hours or more (e.g., 48 or 72 hours) after exposure toUV-A radiation when screening for synergistic combinations of skin-careactives that combat the undesirable effects of UV-A at a dose of, forexample, greater than 5 J/cm² or 10 J/cm². However, when screening forsynergistic combinations of skin-care actives that combat theundesirable metabolic effects of UV-A radiation on fibroblasts at dosesof less than 10 J/cm² or 5 J/cm², time and resource consuming steps inthe screening process may be avoided by not testing at the lower doses.

FIGS. 7A, 7B, 7C, 7D and 7E illustrate the kinetic data obtained bydetecting the extra cellular acidification rate of fibroblasts. Thefibroblasts are obtained in the same way as described above with regardto FIGS. 7A, 7B, 7C, 7D and 7E, and the test was performed according tothe method described in the Test Methods section below. In FIGS. 7A, 7B,7C, 7D and 7E the basal ECAR values are represented by plots 120A, 120B,120C, 120D and 120E; the response ECAR values are represented by plots125A, 125B, 125C, 125D and 125E; and the control ECAR values arerepresented by plots 127A, 127B, 127C, 127D and 127E. The basal values120A, 120B, 120C, 120D and 120E and control values 127A, 127B, 127C,127D and 127E are obtained in the same way as described above. Theresponse ECAR values 125A, 125B, 125C, 125D and 125E correspond to theresponses of the fibroblasts after exposure to 1, 5, 10, 20 or 30 J/cm²of UV-A radiation (365 nm), respectively. As can be seen in FIG. 7A, theresponse ECAR value 125A of the fibroblasts after exposure to 1 J/cm² ofUV-A was generally about the same as the basal value 120A, which mayindicate that the glycolysis metabolic pathway of the fibroblast isrelatively unaffected at this dose. But as illustrated in FIGS. 7B and7C, the response ECAR values 125B and 125C of the fibroblasts afterexposure to 5 and 10 J/cm² of UV-A, respectively, were higher than thecorresponding basal values 120B and 120C. Surprisingly, when thefibroblasts were exposed to 20 J/cm² of UV-A, as illustrated in FIG. 7D,the response ECAR value 125D started out higher than the basal value120D, but appears to decrease until eventually (i.e., approximately 85to 102 minutes) crossing over the basal value 120D line to end up lowerthan the basal value 120D. The response ECAR value 125E of thefibroblasts after exposure to 30 J/cm2 is substantially lower than thebasal value, which may indicate that the glycolysis metabolic pathway ofthe fibroblasts has completely shut down. Thus, it is believed, withoutbeing limited by theory, that the data illustrated in FIGS. 7A, 7B, 7C,7D and 7E indicate that both the time after exposure and dose areimportant factors to consider when providing a suitable way to identifysynergistic combinations of skin-care actives that combat the effects ofUV-A radiation on fibroblast glycolysis. In particular, doses above andbelow 20 J/cm² may have very different effects on the glycolysismetabolism of fibroblasts, and appreciating these differences can helpidentify beneficial combinations of skin-care actives that combat theundesirable effects of UV-A without inhibiting the desirable effects.

FIG. 8 illustrates fibroblast ECAR responses at 24 hours after exposureto UV-A radiation (365 nm). The 24-hour values illustrated in FIG. 8were obtained by exposing the fibroblasts to UV-A radiation and thenincubating the plate at 37° C. and 5% CO₂ for 24 hours. Twenty-fourhours after exposure to UV-A, the cells were analyzed according to theTest Methods below. The 24-hour basal ECAR value 90 is shown at the farleft side of the chart 99. Immediately to the right of the basal value90 is the 24-hour response ECAR value 91 for a 1 J/cm² dose of UV-Aradiation followed by the 24-hour response ECAR value 92 for a 5 J/cm²dose. And at the far right of the chart 99 is the 24-hour response ECARvalue 93 for a 10 J/cm² dose. As illustrated in FIG. 8, the fibroblastsappear to show a reduction in ECAR after 24 hours only at a dose of 10J/cm². Thus, the time after exposure, especially up to 24 hours, may bean important factor to consider when screening for synergisticcombinations of skin-care actives that combat effects of UV-A radiationon fibroblast glycolysis, depending on the dose of UV-A.

Personal Care Compositions and Method of Using the Same

Because of the skin health and/or appearance benefit provided by ahealthy skin cells, it may be desirable to incorporate a synergisticcombination of two or more skin-care actives identified according to themethod herein into a cosmetic composition. That is, it may be desirableto include the skin-care actives as ingredients in the cosmeticcomposition. Such cosmetic compositions may include a dermatologicallyacceptable carrier and a synergistic combination of skin-care activesthat reduce, prevent and/or reverse the undesirable metabolic effects ofoxidative stress on keratinocytes, fibroblasts and/or other types ofcells commonly found in skin (e.g., melanocytes, myocytes, stem cells,sebocytes, neurocytes, and adipocytes). In a particularly suitableexample, the composition may contain a safe and effective amount ofniacinamide and tocoquinone. In this example, the niacinamide may beadded at between 0.001% and 20% (e.g., 0.1-10% or 0.5-5%) and thetocoquinone may be added at between 0.0001% and 20% (e.g., 0.01-10%,0.1%-5%, or 0.5%-3%). It may be desirable to add a combination ofniacinamide and tocoquinone sufficient to improve the oxphos andglycolysis metabolic pathways of fibroblasts exposed to hydrogenperoxide relative to using niacinamide and tocoquinone alone.

In some embodiments, the synergistic combination of skin-care activesmay comprise two or more skin-care actives acting together to providemore than a predicted benefit. For example, a first and second activemay each act on one or both of the oxphos and glycolysis pathways toprovide an increase in overall cellular metabolism that exceeds theexpected additive effect of the actives. In this example, a first activemay provide an improvement to the oxphos pathway of a keratinocyte whenused alone, whereas a second active appears to provide no benefit to theoxphos pathway when used alone. However, when used in combination, thefirst and second actives improve oxphos metabolism more than when eitheris used alone. In another example, a first active may improve the oxphosmetabolism of a fibroblast while a second active improves the glycolysismetabolism of the fibroblast. Continuing with this example, the firstand second actives when used in combination improve oxphos andglycolysis metabolism, thereby improving the overall metabolism of thefibroblast. In still another example, a first active may act to combatthe undesirable metabolic effects of a particular stressor on glycolysiswhile the second active acts to combat the undesirable metabolic effectsof the same stressor on oxphos, thereby reducing, preventing and/orreversing the undesirable effect of the stressor on overall cellularmetabolism more than when either active is used alone.

In some embodiments, a synergistic combination of skin-care active maycomprise two or more skin-care actives that act on different types ofskin cells to provide an improvement to the overall health of the skin.For example, first and second actives that act on the oxphos and/orglycolysis pathway of a different type of cell (e.g., keratinocytes andfibroblasts) may be incorporated into a skin-care composition to providea holistic skin-care benefit. In this example, the first active mayimprove the oxphos pathway of keratinocytes leading to an improvement inthe barrier properties of skin, while the second active improves theglycolysis metabolism of fibroblasts leading to an improvement in skinthickness and/or elasticity.

In some embodiments, the synergistic combination may be two or moreskin-care actives that do not inhibit one another with respect toimproving the metabolism of a cell and/or metabolic pathway. Forexample, a first active may be added to improve oxphos metabolism and asecond active may be added to improve glycolysis metabolism. In thisexample, neither active inhibits the respective metabolic benefitprovided by the other active. This type of synergistic combination isimportant because some skin-care actives that may work well when usedalone may not provide the same kind or amount of metabolic improvementwhen used together, which is undesirable. In another example, a firstand second active may both provide an improvement in oxphos metabolism,which can be the same or different (i.e., one active improves metabolismbetter than the other when the actives are used alone), one of theactives also provides a non-metabolic benefit (e.g., economic or safety)over the other active. For example, one of the actives may be easier tosource, cheaper to purchase, easier to formulate, have regulatoryapproval in a particular country or region that the other does notand/or have less and/or no undesirable side effects at higherconcentrations. Thus, the first and second actives may be used in asynergistic combination to provide a desired level of metabolic benefitand a desired level of non-metabolic benefit.

The cosmetic compositions herein may include one or more optionalingredients of the kind commonly included in the particular cosmeticcompositing being provided. For example, the cosmetic composition mayinclude additional skin-care actives known for regulating and/orimproving the condition of mammalian skin. Nonlimiting examples of suchoptional ingredients include emollients, humectants, vitamins; peptides;and sugar amines. Other optional ingredients include sunscreen actives(or sunscreen agents) and/or ultraviolet light absorbers. In certainembodiments, the cosmetic composition may include a colorant, asurfactant, a film-forming composition, and/or a rheology modifier.Suitable cosmetic compositions herein may be in any one of a variety offorms known in the art, including, for example, an emulsion, lotion,milk, liquid, solid, cream, gel, mouse, ointment, paste, serum, stick,spray, tonic, aerosol, foam, pencil, and the like. The cosmeticcompositions may also be incorporated into shave prep products,including, for example, gels, foams, lotions, and creams, and includeboth aerosol and non-aerosol versions. Other cosmetic compositionsinclude antiperspirant, deodorant, and personal cleaning compositionssuch as soap and shampoo. Nonlimiting examples of cosmetic compositionsand optional ingredients suitable for use therein are described in U.S.Publication No. 2010/0239510 filed by Ha, et al., on Jan. 21, 2010.

Compositions incorporating skin-care actives identified by the novelmethods described herein may be generally prepared according toconventional methods known in the art of making compositions and topicalcompositions. Such methods typically involve mixing of ingredients inone or more steps to a relatively uniform state, with or withoutheating, cooling, application of vacuum, and the like. For example,emulsions may be prepared by first mixing the aqueous phase materialsseparately from the fatty phase materials and then combining the twophases as appropriate to yield the desired continuous phase. In certainembodiments, the compositions may be prepared to provide suitablestability (physical stability, chemical stability, photostability, etc.)and/or delivery of active materials. The composition may be provided ina package sized to store a sufficient amount of the composition for atreatment period. The size, shape, and design of the package may varywidely. Some package examples are described in U.S. Pat. Nos. D570,707;D391,162; D516,436; D535,191; D542,660; D547,193; D547,661; D558,591;D563,221; and U.S. Publication Nos. 2009/0017080; 2007/0205226; and2007/0040306.

The personal care compositions disclosed herein may be applied to theskin at an amount and frequency to improve skin elasticity, improvehydration, regulate or improve skin condition, maintain or improve thesigns of skin aging, or maintain or improve insult-affected keratinoustissue. For example, it may be desirable to identify a target area ofskin in need of a skin-care benefit applying a cosmetically safe andeffective amount of the personal care composition to the target area. Insome instances, the skin-care composition may be used as a specializedtreatment for an entire face, with concentrated usage in areas withexpression lines, wrinkles, undesirable tone or spots. Further, thepersonal-care compositions herein may be used to produce a chronicand/or acute skin or cosmetic benefit by topically applying to the skinof a mammal in need of such treatment a safe and effective amount of theskin-care composition.

Test Method

This method enables the non-lethal, simultaneous detection of metabolicindicators associated with the oxidative phosphorylation and glycolysismetabolic pathways in a controlled environment. The method also enablesthe collection of kinetic data. When assessing the metabolic response toa stressor, test agent or combination of test agents, it is important toassess both metabolic pathways simultaneously to understand how the twometabolic pathways interact with the stressor or test agent(s) and/or toone another. Additionally, it is important to monitor the metabolicpathways in real time (i.e., repeating periodic measurements) to observetrends and/or transient responses that may be missed when using methodsthat provide only static data. Thus, destructive tests are not suitablefor use herein since they typically only allow for a single measurement.

It is also important to detect the metabolic indicator in a controlledenvironment to reduce the likelihood of artifact introduction. Asuitable controlled environment should minimize or prevent anyundesirable influence by external environmental conditions (e.g.,temperature, pressure, humidity, light, and contact by undesirablegaseous, liquid and/or solid contaminants). For example, if themetabolic indicator being detected is oxygen consumption rate and thetest sample is open to the environment, the measured oxygenconcentration may not accurately reflect the amount of oxygen consumedby the cells, since at least some of the consumed oxygen may be replacedby environmental oxygen. Additionally, the test method itself should notintroduce artifact into the measurement. For example, it is known thatthe Clark Electrode consumes oxygen, which is the very thing it issupposed to detect. Thus, the oxygen concentration measured by a ClarkElectrode device may not accurately reflect the amount of oxygenconsumed by the cells in the test.

It is to be appreciated that environmental changes such as a change inthe temperature of the medium may result in unwanted measurement errors.In particular, the capacity of the media to hold dissolved gasseschanges with temperature, and therefore may result in an apparent changein dissolved gas concentration as the media seeks equilibrium with thesurrounding environment. Further, the measurement properties of at leastsome types of sensors may change with temperature. Accordingly, it maybe particularly desirable to control the environmental conditions suchas the temperature of the medium in the test vessel and/or surroundingenvironment or apply a correction factor to the measurement. Similarly,any evaporation from the media due to other uncontrolled environmentalconditions such as humidity or exposure to air currents may artificiallyimpact the measurements made from various sensors including those ofdissolved gases, ions, and temperature. Thus, providing a controlledenvironment to minimize or eliminate these factors can be important.

In some instances, the device used to detect the metabolic parametersmay include a stage adapted to receive and support a test vessel (e.g.,multiwell microplate). The device may also include a plunger configuredto receive a barrier for isolating the environment within the testvessel from the external environment. The barrier may be configured tomechanically cooperate with a portion of the test vessel to seal theopening in the test vessel, for example, by mating with a seat or stepin the test vessel wall. Additionally or alternatively, the plunger andbarrier may be configured to reduce and/or expand the volume of the testvessel and the volume of the medium within the test vessel including atleast a portion of the cells (e.g., 5-50%). For example, the barrier maybe inserted into and/or retracted out of the test vessel by relativemovement of the stage and the plunger. In some embodiments, the methodmay include perfusing additional media through the vessel and/orreplenishing the medium. Reducing the volume of the medium enables thetemporary creation of a highly concentrated volume of cells within alarger volume of cell media to improve the ability of the sensor(s) todetect sensitive changes in metabolic indicators in the medium thatresult from biological activity of the cells. By temporarily, ratherthan permanently, reducing the media volume (and therefore concentratingthe cell/media mixture), cells are exposed to a non-normal environmentfor only a brief period of time, and therefore may not be adverselyaffected (e.g., killed) by the measurement process.

The instrument should also include a sensor capable of analyzing thedesired metabolic indicator(s). In some embodiments, the sensor may bedisposed on the barrier and/or plunger. The sensor should be in sensingcommunication with the medium and configured to detect the desiredmetabolic indicator. The sensor may be configured to sense the presenceand/or the concentration of the metabolic indicator; sense a rate ofchange of a concentration of the metabolic indicator; and/or sense afirst concentration of a first metabolic indicator, sense a secondconcentration of a second metabolic indicator, and determine arelationship between the first concentration and the secondconcentration. It may be desirable to configure the sensor to detect themetabolic parameter without disturbing the cells. The instrument mayalso include a computer programmed to automate one or more aspects ofthe tests, including data collection and recording, cycling through oneor more test steps and transferring a test agent or stressor to theextracellular environment. In some embodiments, the sensor may becoupled to the computer.

Suitable nonlimiting examples of devices that provide a controlledenvironment; non-lethal and simultaneous detection of metabolicindicators; and kintetic data are disclosed in U.S. Pat. Nos. 7,276,351;7,638,321; and 7,851,201, to Teich et al.; and U.S. Pat. No. 8,202,702to Neilson, et al. A particularly suitable device is the XFExtracellular Flux Analyzer available from Seahorse Bioscience,Massachusetts.

Cell Culture and Sample Preparation

Cells to be tested according to the method herein may be obtained by anysuitable means known in the art. For example, the cells may be isolatedfrom a natural environment (e.g., the skin of a person) or purchasedfrom a suitable commercial source. The cells may be a primary cell line(i.e., a cell line that is isolated from a biological tissue source andpropagated under normal tissue culture conditions) or an immortalizedcell line (i.e., a cell line that has been modified via chemical orgenetic modification such that its proliferation and doubling index aresignificantly greater than that available from primary cell lines). Insome embodiments, frozen primary or immortalized cell lines may beobtained from a suitable commercial source and diluted with anappropriate growth medium into tissue culture flasks (e.g., collagencoated T-75 flask available from BD Biosciences) for incubation (e.g.,at 37° C.). Once the cells are ready for testing, they may be placed ina suitable test vessel (e.g., multi-well vessels, single-well vessels,one or more tubes, and conventional test plates such as a 12-well plate,24-well plate, 96-well plate or the like). A test medium may be providedin the test vessel to form an extracellular environment. The test mediumshould keep the cells alive and healthy for at least the duration of thetest (e.g., at least 4 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72hours). The number of cells in each well should be sufficient to obtaina suitable measurement (e.g., 4×10⁴ for keratinocytes and 1×10⁵ forfibroblasts). It is to be appreciated that during testing the cells maybe suspended in the test medium, attached to a suitable substratedisposed in the test vessel and/or attached to the test vessel.

By way of example, the keratinocytes discussed above with regard toFIGS. 1A, 1B, 1C, 2, 3A, 3B, 3C and 4 are frozen, human primarykeratinoctyes obtained from Gibco Life Sciences. The keratinocytes weregrown to 70-80% confluence in EpiLife® brand keratinoyte medium(available from Invitrogen, Grand Island, N.Y.) supplemented with humankeratinocyte growth supplement and gentamicin/amphotericin B ×500solution, both available from Invitrogen. For each test, two separatedonors of keratinocytes were cultured and equal cell numbers of eachdonor were combined in the test vessel. In this example, 2×10⁴keratinocytes from each donor were placed in a Gelatin-coated, 24-wellplate, for a total of approximately 4×10⁴ keratinocytes in each well.The number of cells present may be determined by any suitable meansknown in the art (e.g., using a Coulter counter). The keratinocytes wereplaced in the Gelatin-coated plates 24 hours prior to testing and 100 μLof the keratinocyte medium described above was added to each well. Atest medium was made by modifying a commercially available EpiLife®medium that is devoid of buffers, calcium, glucose, pyruvate, andglutamine. The EpiLife® medium was modified by adding 10 ng/ml Insulin;10 ng/ml Hydrocortisone; 60 μM Calcium Chloride; 10 mM of Glucose; 1 mMof Pyruvate; and 2 mM of Glutamine. The test medium was warmed to 37° C.and the pH adjusted to 7.4 with sodium hydroxide. The keratinocytemedium in the wells was replaced by the test medium 1 hour prior totaking measurements

With regard to the fibroblasts discussed above in FIGS. 5A, 5B, SC, 5D,5E, 6, 7A, 7B, 7C, 7D, 7E and 8 frozen, human dermal fibroblasts wereobtained from the BJ cell line commercially available from ATCC,Bethesda, Md. The fibroblasts were grown to 70-80% confluence in afibroblast culture medium of Eagle's Minimum Essential Medium (“EMEM”)supplemented with 10% fetal bovine serum (“FBS”) andgentamicin/amphotericin B ×500 solution (EMEM and FBS are available fromATCC and gentamicin/amphotericin B ×500 solution is available fromInvitrogen). The fibroblasts were plated at 1×10⁵ per well inGelatin-coated plates 24 hours prior to testing. Each well included 100μL of the fibroblast medium described above. A test medium was made fromDulbecco's Modified Eagle Medium (available from Seahorse Bioscience),25 mM Glucose and 1 mM pyruvate. The test medium was warmed to 37° C.and the pH adjusted to 7.4. The cells were grown and plated at 37° C.and 5% CO₂ air. The fibroblast medium in the wells was replaced by thetest medium 1 hour prior to taking measurements.

The Gelatin-coated plates may be prepared by coating each well in amulti-well plate (e.g., 24-well or 96-well plates available fromSeahorse Biosciences, Massachusetts) with 0.2% Gelatin brand proteinsolution (available from Sigma) diluted with phosphate buffered saline(“PBS”). The gelatin is diluted 1:10 to 0.2% in sterile PBS at 37° C.,and then 50 uL of the diluted gelatin solution is added to each well andincubated at room temperature for 30 minutes. Excess liquid is removedby aspiration without touching the wells and the surface is allowed todry for at least 2 hours.

Exposing Cells to a Stressor or Test Agent

The cells may be exposed to a stressor or test agent by any suitablemeans known in the art. In some embodiments, the cells may be exposed tothe stressor prior to placing the cells in the instrument that will beused to detect the metabolic parameter. For example, the cells may beexposed to UV radiation using a BIO-SUN brand solar simulator (availablefrom Vilber Lourmat, France) prior to placing the cells in an XFExtracellular Flux Analyzer. In this example, the test medium may beremoved from plate wells and replaced with 100 uL of PBS to reduce anyundesirable effects the test medium may have on the UV radiation (e.g.,absorbance or reflectance). In embodiments where a metabolic indicatorwill not detected within a suitable time after exposure (e.g., whenobtaining a 24-hour measurement), the PBS may be replaced with theappropriate medium (e.g., the keratinocyte or fibroblast mediumdescribed above) immediately after exposure to the stressor but prior toplacing the cells in an incubator. In order to obtain data fromnon-irradiated cells, the portion of the plate that will contain thenon-irradiated cells may be covered with a UV impervious material suchas aluminum foil. In some instances, a stressor and/or a test agent maybe introduced into the test vessel before and/or after the cells areplaced in the test device. For example, the test device may include oneor more injector ports that enable the introduction of a substancedirectly into the test vessel before, during and/or after testing.

Measurement of Oxidative Phosphorylation and Glycolysis MetabolicIndicators

Oxygen consumption rate and extracellular acidification rate may bedetected using an XF Extracellular Flux Analyzer or equivalent. Thedevice should be capable of non-lethally and simultaneously detectingmetabolic indicators of the oxphos and glycolysis pathways in acontrolled environment, as well as providing kintetic data. The cellsmay be provided in a multi-well plate suitable for use with theinstrument (e.g., 24-well or 96 well plate) and washed prior to testing.The cells may be washed by any suitable means known in the art (e.g.,using a Seahorse Biosciences XF prep station). It may be desirable touse a suitable prep station such as an XF prep station available fromSeahorse Biosciences). When washing the cells, it may be desirable toremove the medium from the wells and wash the cells three times with asuitable amount of test medium (e.g., 180 μL in a 96-well plate or 600μL in a 24-well plate). After washing the cells, a suitable amount(e.g., 180 μL) of an appropriate test medium is placed in each well, andthe cells are equilibrated at 37° C. in a CO₂-free incubator for 1-1.5hours prior to taking placing the plate in the instrument for testing.Following the equilibration period, load the plate containing the cellsinto the instrument and equilibrate according to manufacturer'sinstructions. The entire test is conducted at 37° C. In someembodiments, the instrument may be to provide a three minute mix cycle,a two minute wait cycle, and a 3 minute measurement cycle forkeratinocytes and a two minute mix cycle, two minute wait cycle, and 3minute measurement cycle for fibroblasts. The cycles should be repeatedfor at least 88 minutes. It is to be appreciated that the cycles andtimes may be modified according to cell type and experiment design, asdesired.

Example

The following example illustrates how the method herein can be used toidentify synergistic combinations of skin care actives. Hydrogenperoxide is a well known ROS, and is used in this example to illustratethe undesirable metabolic effects associated with exposure of a cell toan oxidative stressor. The hydrogen peroxide was prepared at 10X workingconcentration in the fibroblast test medium described above. The testagents are niacinamide (also known as vitamin B₃) and tocoquinone bothcommercially available from Sigma. The test agents were each prepared at10× the final working concentration in the fibroblast test mediumdescribed above to provide a test agent solution. The stressor solutionand the test agent solutions were both warmed to 37° C. and pH adjustedto 7.4. The cells are frozen, human, dermal fibroblasts obtained fromATCC (BJ cell line). The cells were cultured and prepared according tothe test method described above. The fibroblasts were plated inGelatin-coated, 96-well plate. Each well contained approximately 4×10⁴cells and 180 μL of fibroblast test medium (i.e., Seahorse BiosciencesDMEM, 25 mM glucose and 1 mM pyruvate) at 37° C. and pH 7.4.

An XF Extracellular Flux Analyzer was used to detect the metabolicindicators corresponding to the glycolysis and oxphos metabolic pathways(i.e., extracellular acidification rate and oxygen consumption rate).The 96-well plate was loaded into the analyzer and equilibratedaccording to the manufacturer's instructions. The test agents andstressor were each loaded into an automated injection port of the XFcartridge plate. The analyzer was programmed to sequentially run a twominute mix cycle and a 4 minute measurement cycle continuously for atleast 108 minutes. Data points were collected and recorded by theanalyzer. The analyzer was allowed to complete three cycles prior to theaddition of the stressor solution or test agent solutions to provide abasal value for each metabolic indicator. After the third cycle wascompleted (i.e., about 18 minutes after detection began), the stressorand test agents were added to the wells from the appropriate injectionport. The stressor solution was added to the well in sufficient amountto provide 1.5 mM hydrogen peroxide in the well. The test agents wereadded in sufficient amount to provide 0.25 mM niacinamide and 0.025 mMtocoquinone in the well. Eight wells in the 96-well plate were used as acontrol. The control the fibroblasts and fibroblast medium but was notcontacted with hydrogen peroxide or either test agent. The fibroblastswere subjected to four test conditions. Each test condition used 8 wellsof the 96-well plate. In the first test condition, the fibroblasts werecontacted with the only 1.5 mM hydrogen peroxide. In the second testcondition, the fibroblasts were with 0.25 mM niacinamide and 1.5 mMhydrogen peroxide in combination. In the third test condition, thefibroblasts were contacted with 0.025 mM tocoquinone and 1.5 mM hydrogenperoxide in combination. In the fourth test condition, the fibroblastswere contacted with 0.25 niacinamide, 0.025 mM tocoquinone and 1.5 mMhydrogen peroxide in combination. The results are illustrated in Table 1and Table 2 below and FIGS. 9 and 10.

TABLE 1 Glycolysis Average ECAR value per treatment group (percentage ofrate 3) 0.25 mM niacinamide + 0.25 mM 0.025 mM 0.025 mM niacinamide +tocoquinone + tocoquinone + Rate number 1.5 mM hydrogen 1.5 mM hydrogen1.5 mM hydrogen 1.5 mM hydrogen (time point) Control peroxide peroxideperoxide peroxide 1 110.46 101.82 107.35 107.45 103.25 2 101.32 100.6699.41 99.58 100.64 3 100 100 100 100 100 4 120.82 48.46 64.35 56.7758.78 5 108.27 29.55 33.18 35.13 35.31 6 110.45 33.64 34.48 32.57 32.697 111.48 37.41 42.19 32.46 32.29 8 112.20 28.20 49.60 34.54 34.67 9111.60 17.74 55.87 36.55 38.49 10 110.74 15.41 60.52 36.68 42.70 11110.90 14.87 62.89 35.75 47.52 12 112.18 13.82 60.57 32.86 55.00 13113.44 13.32 57.46 29.78 58.82 14 114.71 12.78 55.04 28.15 59.70 15115.79 11.82 52.85 28.07 61.66 16 117.40 11.52 51.52 30.73 65.94 17118.38 11.36 49.85 31.49 69.54 18 118.98 11.57 47.58 31.18 70.74

Table 1 illustrates the effects of hydrogen peroxide, niacinamide andtocoquinone, alone and in combination, on the acidification rate of theextracellular environment of fibroblasts. The data from Table 1 wasanalyzed via XF Software Version 1.8 to calculate the averages for eachtreatment group at each time point and compared to the change frombaseline as a percentage that is represented graphically in FIG. 9. Asdiscussed above, the first three measurements were made prior to theaddition of the stressor or test agents to provide a basal value. FIG. 9provides a plot for each of the control 110, 1.5 mM H₂O₂ alone 111, 0.25mM niacinamide and 1.5 mM H₂O₂ in combination 113, 0.025 mM tocoquinoneand 1.5 mM hydrogen peroxide in combination 112, and 0.25 mMniacinamide, 0.025 mM tocoquinone and 1.5 mM hydrogen peroxide incombination 114. As illustrated in Table 1 and FIG. 9, the 1.5 mMhydrogen peroxide by itself appears to causes a substantial decrease inextracellular acidification rate. After the sharp drop, theacidification rate continues to decrease until eventually it falls belowthe basal value approximately 40 minutes after addition of the hydrogenperoxide. Thus, a reasonable conclusion may be drawn that hydrogenperoxide causes a decrease in the glycolysis metabolism in humanfibroblasts. Niacinamide, when added by itself at 0.25 mM, appears tocause an initial decrease in acidification rate before returning to thebasal level or slightly above within about 20 minutes after its additionto the well. From this, a reasonable conclusion can be drawn that 0.25mM niacinamide does not significantly affect glycolysis metabolism.However, when added in combination with hydrogen peroxide, niacinamideappears to lessen the detrimental effect of the hydrogen peroxide onglycolysis metabolism. That is, both the 0.1 mM and 0.25 mM niacinamideconcentrations appear to slow the decrease in acidification rate andprevent the acidification rate from falling as far below the basal levelwhen hydrogen peroxide is added alone.

TABLE 2 Oxidative Phosphorylation Average OCR value per treatment group0.25 mM niacinamide + 0.25 mM 0.025 mM 0.025 mM niacianamide +tocoquinone + tocoquinone + Rate number 1.5 mM hydrogen 1.5 mM hydrogen1.5 mM hydrogen 1.5 mM hydrogen (time point) Control peroxide peroxideperoxide peroxide 1 99.67 97.05 99.16 101.04 99.17 2 101.37 99.80 101.76101.44 102.02 3 100 100 100 100 100 4 88.75 −139.75 −21.15 −21.48 38.285 93.67 −4.58 63.31 64.55 106.15 6 94.26 −6.07 60.15 68.43 103.02 793.874 −24.10 47.41 59.47 89.81 8 93.35 −34.10 38.52 53.35 79.85 9 92.32−44.54 34.85 49.83 75.55 10 91.57 −53.81 32.39 48.18 72.23 11 90.66−58.25 31.10 48.82 69.38 12 89.49 −63.85 32.43 49.81 70.67 13 88.58−70.69 30.49 47.50 70.85 14 87.63 −73.38 29.48 44.97 70.80 15 86.72−76.46 28.18 41.00 71.42 16 86.37 −76.97 26.95 37.55 71.60 17 85.64−76.26 25.83 32.67 72.94 18 84.88 −76.76 23.41 28.15 72.89

Table 2 illustrates the effects of hydrogen peroxide, niacinamide andtocoquinone, alone and in combination, on the oxygen consumption rate offibroblasts. The data from Table 2 was analyzed via XF Software Version1.8 to calculate the averages for each treatment group at each timepoint and compared to the change from baseline as a percentage that isrepresented graphically in FIG. 10. As discussed above, the first threemeasurements were made prior to the addition of the stressor or testagents to provide a basal value. FIG. 10 provides a plot for each of thecontrol 210, 1.5 mM H₂O₂ alone 211, 0.25 mM niacinamide and 1.5 mM H₂O₂in combination 213, 0.025 mM tocoquinone and 1.5 mM H₂O₂ in combination212, and 0.25 mM niacinamide, 0.025 mM tocoquinone and 1.5 mM hydrogenperoxide in combination 214. As illustrated in Table 2 and FIG. 10, 1.5mM H₂O₂ by itself initially causes a relatively sharp decrease in OCRfollowed by a less rapid increase. About 20 minutes or so after addingthe hydrogen peroxide, the OCR began to approach a relatively constantrate, which is much lower than the basal rate. Thus, a reasonableconclusion may be drawn that hydrogen peroxide causes a decrease in theoxphos metabolism of human fibroblasts. As illustrated in FIG. 10, theniacinamide, when added at 0.25 mM in combination with the H₂O₂,generally tracks the shape of the hydrogen peroxide plot 211, butappears to lessen the reduction in OCR caused by the hydrogen peroxide.The response from tocoquinone, when added at 0.025 mM in combinationwith the H₂O₂, appears to lessen the reduction in OCR caused by thehydrogen peroxide about the same or slightly less than the niacinamide.It is important to note that neither the niacinamide nor the tocoquinonein this example, when combined individually with the H₂O₂, were able tobring the OCR of the fibroblasts back up to the basal value.Surprisingly, as illustrated in FIG. 10, when the niacinamide andtocoquinone are both used in combination with the H₂O₂, the OCR of thefibroblasts is brought back to about the basal value. Thus, in thisexample, the combination of niacinamide and tocoquinone provides asynergistic response to the reduction in oxphos metabolism offibroblasts caused by hydrogen peroxide.

The dimensions and values disclosed herein are not to be understood asbeing strictly limited to the exact numerical values recited. Instead,unless otherwise specified, each such dimension is intended to mean boththe recited value and a functionally equivalent range surrounding thatvalue. For example, a dimension disclosed as “40 mm” is intended to mean“about 40 mm” Additionally, properties described herein may include oneor more ranges of values. It is to be understood that these rangesinclude every value within the range, even though the individual valuesin the range may not be expressly disclosed.

All documents cited in the Detailed Description of the Invention are, inrelevant part, incorporated herein by reference; the citation of anydocument is not to be construed as an admission that it is prior artwith respect to the present invention. In particular, U.S. ProvisionalApplication Ser. Nos. 61/711,500 and 61/711,521 are incorporated hereinby reference in their entirety. To the extent that any meaning ordefinition of a term in this document conflicts with any meaning ordefinition of the same term in a document incorporated by reference, themeaning or definition assigned to that term in this document shallgovern.

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to those skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

What is claimed is:
 1. A method of identifying or evaluating asynergistic combination of actives for use in a personal carecomposition, the method comprising: a. providing first, second and thirdtest vessels, each test vessel comprising cells disposed in a medium; b.contacting the cells in the first test vessel with a first test agent;c. non-lethally detecting a metabolic indicator associated with each ofglycolysis and oxidative phosphorylation to provide a response of eachmetabolic pathway to the first test agent; d. contacting the cells inthe second test vessel with a second test agent; e. non-lethallydetecting a metabolic indicator associated with each of glycolysis andoxidative phosphorylation to provide a response of each metabolicpathway to the second test agent; f. contacting the cells in the thirdtest vessel with the first and second test agents in combination; g.non-lethally detecting a metabolic indicator associated with each ofglycolysis and oxidative phosphorylation to provide a response of eachmetabolic pathway to the first and second test agents in combination;and h. identifying the combination of the first and second compositionsas a synergistic combination of actives when at least one of theresponses in (g) indicates a synergistic improvement over thecorresponding response in (c) or (e).
 2. The method of claim 1, whereinthe improvement corresponds to an additive effect or more than additiveeffect.
 3. The method of claim 1, further comprising exposing the cellsin at least one of the first, second and third test vessels to anoxidative stressor or ROS prior to contacting the cells with the firstor second test agent.
 4. The method of claim 3, wherein the metabolicindicators are detected at least 1 hour after exposure to the stressoror ROS.
 5. The method of claim 3, wherein the stressor is selected fromthe group consisting of ultraviolet radiation, cigarette smoke, ozone,engine exhaust, diesel exhaust, smog, surfactants, and radiation from acomputer monitor or television.
 6. The method of claim 5, wherein thecells are selected from the group consisting of keratinocytes andfibroblasts and the stressor is selected from the group consisting ofUV-B and UV-A radiation.
 7. A method of making a personal carecomposition that provides a skin health benefit and is suitable fortopical application to skin, the method comprising: a. identifying asynergistic combination of actives according to the method of claim 1;and b. incorporating a safe and effective amount of the actives into apharmaceutically acceptable carrier.
 8. A method of improving skinhealth, comprising: a. identifying a target area of skin in need of askin-care benefit; and b. applying a cosmetically safe and effectiveamount of a personal care composition made according to the method ofclaim 14 to the target area.
 9. A method of identifying or evaluating asynergistic combination of actives, comprising: a. providing a firsttest vessel comprising a first type of cells disposed in a first mediumand a second test vessel comprising a second type of cells disposed in asecond medium; b. providing a basal value for each of glycolysis andoxidative phosphorylation for the first type of cells and the secondtype of cells; c. contacting the first and second types of cells with afirst test agent and a second test agent in combination; d. non-lethallydetecting a metabolic indicator associated with each of glycolysis andoxidative phosphorylation in the first and second types of cells toprovide a response of each metabolic pathway to the first and secondtest agents in combination; and e. identifying the combination of thefirst and second test agents as a synergistic combination of activeswhen at least one of the responses indicates a synergistic improvementover the corresponding basal value.
 10. The method of claim 9, furthercomprising exposing the first and second types of cells to an oxidativestressor or ROS.
 11. The method of claim 10, wherein the cells areexposed to the oxidative stressor or ROS prior to contacting the cellswith the test agents.
 12. The method of claim 9, wherein one of thefirst and second types of cells are selected from the group consistingof keratinocytes and fibroblasts.
 13. The method of claim 9, whereindetection of the metabolic indicators is done in a controlledenvironment.
 14. The method of claim 9, wherein the metabolic indicatorassociated with the oxidative phosphorylation pathway is detected bymeasuring at least one of oxygen consumption rate and carbon dioxidegeneration rate.
 15. The method of claim 9, wherein the metabolicindicator associated with the glycolysis pathway is detected bymeasuring at least one of extracellular acidification rate,extracellular lactic acid rate and lactate concentration rate.
 16. Amethod of making a personal care composition that provides a skin healthbenefit and is suitable for topical application to skin, the methodcomprising: a. identifying a beneficial combination of actives accordingto the method of claim 9; and b. incorporating a safe and effectiveamount of the actives into a pharmaceutically acceptable carrier.
 17. Amethod of improving skin health, comprising: a. identifying a targetarea of skin in need of a skin-care benefit; and b. applying acosmetically effective amount of a personal care composition madeaccording to the method of claim 16 to the target area.
 18. A personalcare composition comprising a synergistic combination of skin-careactives, the composition comprising: a. a dermatologically acceptablecarrier; b. a safe and effective amount of niacinamide as a firstactive; and c. a safe and effective amount of tocoquinone as a secondactive, wherein the safe and effective amount of niacinamide andtocoquinone in combination improves at least one of glycolysis andoxidative phosphorylation metabolism of a skin cell suffering fromoxidative stress as a result of exposure to a stressor and provides asynergistic advantage over using the actives alone.
 19. The compositionof claim 18, wherein the oxidative stressor is selected from ultravioletradiation, cigarette smoke, ozone, engine exhaust, diesel exhaust, smog,surfactants, and radiation from a computer monitor or television. 20.The composition of claim 18, wherein at least one of actives in thesynergistic combination provides a non-metabolic benefit.